Cloning And Expression Of Endogenous Cellulase Genes From Odontotermes Formosanus (Isoptera: Termitidae)

In nature, degradation of cellulose is performed by mixtures of hydrolytic enzymes collectively known as cellulases. The cellulases are divided into three major groups: endoglucanases (EG, EC 3.2.1.4), cellobiohydrolases (exoglucanases, CBH, EC 3.2.1.91), andβ-glucosidases (BG, EC 3.2.1.21). The study of cellulose digestion in insects is most advanced in termites, which are classified into lower termites, which possess large populations of cellulolytic protozoa in the hindgut, and higher termites, which do not have cellulolytic symbiotes in the gut. The endogenous cellulolytic enzymes of termites primarily consist of endo-P-1,4-glucanase andβ-glucosidase.In this study, we report the cloning, expression and analysis of the distribution and enzyme activity of the cellulases from the termite, Odontotermes formosanus.1 Cloning of cellulase genesWith 5'-and 3'-RACE method, we obtain two complete cellulase cDNAs from 0. formosanus. Blasting on the NCBI with the deduced proteins, we found many cellusase sequences from other termites. The endo-β-1,4-glucanase andβ-glucosidase sequence presented more than 75% and 52% identity with other termite cellulases, respectively.2 Recombinant expression of cellulase genes in Escherichia coliThe cDNAs of two cellulases were transformed into the expression vector of pET-28a, and the recombinant vector was transformed into E. coli of BL21.Two distinctive bands was observed between 45.0 and 66.2 kDa, and western blot analysis using anti His-tag antibody confirmed that the inserted two cellulase cDNAs was overexpressed in E. coli. Unfortunately, no cellulase activity was detected in the recombinant proteins. 3 Recombinant expression of cellulase genes in insect cellsThe two cellulase genes were expressed in insect cells using Baculovirus Expression Vector System (BEVS). The recombinant proteins were present as a single band by SDS-PAGE, and the consistent result of Western blot using polyclonal antiserum confirmed that the inserted cellulase cDNAs were expressed in insect cells. The results of enzyme activity assay showed that, the activities of endo-β-1, 4-glucanase andβ-glucosidase were 0.190 and 0.117U, respectively.4 Variations of the transcripts and expression levels of cellulases in tissues and castesWe conducted RT-PCR experiments using cDNAs prepared from different castes and worker tissues. And by the analysis of SDS-PAGE using total protein extracts from different castes and worker tissues and Western blot using polyclonal antiserum, we investigated the transcript variations of two cellulases. At last, we examined the enzyme activity of two cellulases in different casts and worker tissues. We found that the two endogenous cellulases were excreted by salivary glands and midgut epithelia.